The MO-MDSC fraction of BW-Sp3, but not EG7, tumor–bearing mice gives rise to macrophages able to suppress polyclonal T-cell activation via NO. (A) Total CD11b⁺Gr-1⁺ MDSCs were purified from BW-Sp3 or EG7 tumor-bearers and added in various amounts to naive AKR or C57BL/6 splenocytes, respectively. These cocultures were stimulated with 1 μg/mL anti-CD3, either following the “short-term culture” or “long-term culture” protocol (“Methods”). (B) BW-Sp3 and EG7 MO- and PMN-MDSC fractions were purified and added in the indicated amounts to naive AKR or C57BL/6 splenocytes, respectively. The experiment was executed according to the long-term culture protocol. (C) Supernatant was collected at the end of the culture and nitrite concentrations were determined. (D) Purified BW-Sp3 MO-MDSCs were added at a 1:1 ratio to naive AKR splenocytes and stimulated following the long-term culture protocol in the presence or absence of the indicated inhibitors. (E) At the end of the long-term culture, plastic nonadherent cells were thoroughly washed away and pictures from the adherent cells were taken. Adherent cells were collected by gentle scraping, and expression of the indicated markers was analyzed by flow cytometry. The percentage of CD11b⁺F4/80⁺ mature macrophages (gate R1) in each adherent cell population is indicated. Histograms represent gated CD11b⁺ cells stained with isotype control mAbs (dotted line) or marker-specific mAbs (full line). (F) Purified BW-Sp3 MO-MDSCs were added at different ratios to naive AKR splenocytes and cultured for 3 days. Subsequently, anti-CD3 was added to either undisturbed cultures (= total: adherent + nonadherent cells) or to the plastic nonadherent fraction of the cultures, which was transferred into fresh wells (= nonadherent cells). Twenty-four hours later, ³H-thymidine was provided and was allowed to incorporate for another 18 hours. *P < .05; **P < .01; NS indicates not significant. One representative experiment of at least 3 is shown.